High-performance liquid chromatography (hereafter referred to as “HPLC”) is known as a chemical separation method utilizing a liquid flow system. HPLC can be used for various types of chemical analysis, and various types of HPLC apparatus have been developed according to the applications involved. One such apparatus is a glycosylated hemoglobin measuring apparatus which is used to diagnose diabetes. This measuring apparatus uses blood as a sample, and generally measures the proportion of hemoglobin A1c (hereafter referred to as “HbA1c”) relative to the total amount of hemoglobin contained in the blood. In concrete terms, in this apparatus, a sample solution is prepared by diluting blood with an appropriate diluent, and respective hemoglobin components such as HbA1c and the like contained in the sample solution are developed inside a column using an eluant. As a result, the respective hemoglobin components contained in the sample solution are eluted from the column as separated from each other. The absorbance of the eluate that flows out of the column is constantly measured by a detector which is installed downstream from the column. The absolute values of the HbA1c and other hemoglobins that are eluted from the column are determined on the basis of the absorbance thus measured. Furthermore, the apparatus finally calculates the proportion of HbA1c relative to the total amount of hemoglobin including the HbA1c.
However, in the glycosylated hemoglobin measuring device consisting of a conventional HPLC apparatus, the measured value of the HbA1c fluctuates according to the dilution rate of the blood. In the case of HbA1c originating from the same blood, it would be expected that the measurement results in the same proportion for the HbA1c regardless of the dilution rate of the blood, i.e., regardless of the blood concentration in the sample. In reality, however, the measured value differs according to the blood concentration, so that a fixed value cannot be obtained.
The cause of this problem is thought to be as follows. In cases where the concentrations of the hemoglobin components contained in the eluate varies with the passage of time, the concentration distribution in becomes non-uniform in the radial cross section of the flow path of the eluate that flows through the measurement flow path of the detector. In the piping that extends from the outlet of the column to the measurement flow path of the detector, the eluate is subjected to resistance from the wall surfaces of the piping. As a result, the flow velocity of the eluate becomes slower in the peripheral portions of the cross section of the piping than in the central portion. This state is also maintained inside the measurement flow path. Accordingly, in cases where the hemoglobin concentration in the eluate increases with the passage of time, the progress of liquid substitution is slower in the vicinity of the wall surfaces of the measurement flow path than in the cross-sectionally central portion of the flow path, so that a lower hemoglobin concentration tends to be maintained. As a result, the eluate that flows through the measurement flow path has a concentration gradient that drops from the cross-sectionally central portion of the flow path toward the cross-sectionally peripheral portions. When the absorbance of the eluate inside the measurement flow path is measured in a state in which such a concentration gradient is formed inside the measurement flow path, a value that is lower than the value that should be measured in an ideal state of the eluate in which no concentration gradient is formed is actually measured.
The deleterious effects arising from such laminar flow are especially severe in cases where an extreme variation is seen in the concentration of the eluate over time. Accordingly, in the measurement of glycosylated hemoglobin, the measurement of hemoglobin A0 (hereafter referred to as “HbA0”), which assumes the major portion of the hemoglobin in the blood, and which is the hemoglobin component that is eluted most slowly from the column, is most affected. The effect on HbA0, which has a high concentration in the blood, is much greater than the effect on other hemoglobins such as HbA1c and the like, which have a low concentration. The apparent reason for this is that if the amount of HbA0 present in the largest amounts is measured as a value that is smaller than the true value, then the overall amount of hemoglobin that is present will be estimated on the low side. As a result, the ratio of HbA1c that is present will be calculated on the high side. This view agrees with the experimental rule that the measured value of HbA1c increases with an increase in the concentration of the sample.
If a sufficiently long time is taken for separation by means of a column, the rate of variation in the HbA0 concentration is reduced. Accordingly, the effects of the laminar flow can be alleviated. In this case, however, the separation peaks originating from the respective hemoglobin components tend to overlap in the chromatogram, which is undesirable. Furthermore, such a process is also undesirable in that more time and a greater amount of eluant are required. Especially in the case of a glycosylated hemoglobin measuring apparatus utilizing HPLC, a shortening of the measurement time is desirable. Accordingly, the expenditure of a considerable amount of time on separation by means of a column conflicts with this requirement, and is undesirable.
In the past, a glycosylated hemoglobin measuring apparatus equipped with a diffusion coil has been proposed in order to alleviate the above-mentioned problems occurring inside the measurement flow path of the detector. This diffusion coil is a helical pipe, and is disposed in a position located near the detector in which the measurement flow path is formed. This diffusion coil generates a convection current inside the eluate from the column. As a result, the hemoglobin contained in the eluate is positively diffused in three dimensions. As a result of the diffusing action caused by such a convection current inside the diffusion coil, the concentration gradient in the flow path cross section of the eluate flowing through the measurement flow path is alleviated, so that the measured value of the HbA0 is stabilized to a constant value.
However, although the measured value of the high-concentration component HbA0 can be stabilized to a constant value in the case of a conventional glycosylated hemoglobin measuring apparatus using a diffusion coil, such an apparatus suffers from the following problems. First, since a dilute solution which is by nature relatively immune to the effects of laminar flow is also affected by the convection effect of the diffusion coil, peaks originating from low-concentration hemoglobin components are blunted. Such low-concentration hemoglobin components include HbA1c. As a result, the analytical performance of the apparatus as a glycosylated hemoglobin measuring apparatus drops. Secondly, if a diffusion coil is used, the eluate is subjected to a convection effect before the eluate flows into the measurement flow path. Accordingly, in the eluate that has flowed into the measurement flow path, the hemoglobin components are diffused to a considerable extent not only in the radial direction, but also in the flow direction. As a result of this diffusion in the flow direction, the degree of separation of components that have already once been separated by the column is reduced in the piping that follows the column. As a result, the half-value widths of the peaks originating from the respective components are broadened in the chromatogram, so that the analysis time is increased beyond the conventional value relative to the time required for separation.